Current models of gene regulation suggest that interphase chromosomes are divided into large, functionally distinct, chromatin domains, facilitating independent regulation of individual gene loci. Typically these domains are tens to hundreds of kbp in size. The actual structural and molecular mechanisms underlying the establishment of these domains, however, have remained poorly understood. To date, only indirect structural assays have been available to analyze the chromatin structure of these domains. A key limitation has been the technical difficulty associated with directly visualizing the large-scale chromatin folding surrounding specific genes. Our long term objectives are to dissect the large-scale chromatin folding of specific gene loci, to analyze the cis and trans determinants of these folding motifs, and to determine the functional significance of this level of chromatin organization with regard to transcriptional regulation. Our specific aims for this project period will be to address the following questions: 1. What changes in large-scale chromatin organization accompany transcriptional activation? 2. Do SAR/MAR sequences have a direct, structural role in organizing chromatin domains? 3. What is the relationship between domain boundary elements, defined by molecular assays, and sequences associated with cytologically defined domain boundaries? 4. Do LCR sequences alter large-scale chromatin organization? Using novel visualization methods, in vivo dynamics of specific gene loci will be observed directly and light and electron microscopy will be applied to visualize the large-scale chromatin organization and nuclear positioning of these chromosomal regions. These visualization capabilities will allow direct testing of several structural based models of chromatin domain organization and gene regulation. Moreover, these studies are likely to lay the foundation for future mechanistic studies aimed at dissecting the role of cis and trans determinants of chromatin domain structure and function. Insight acquired from these studies should be of help in guiding the design of future constructs and artificial chromosomes used in gene therapy.